Chung-Seog Song

Regulation of Androgen Action

Publisher Summary This chapter describes the different aspects of regulation of androgen action. Androgens belong to a class of C-19 steroids secreted primarily by the testis and adrenal cortex. Hormonally active androgens promote reproductive and anabolic functions. Both reproductive and anabolic effects of androgens are mediated by their interaction with the androgen receptor (AR), a member of the steroid-thyroid hormone-retinoid-vitamin D superfamily of nuclear receptors (NRs) that function as ligand-activated transcription factors. Almost all of the androgen functions, except its conversion to estrogen by the enzyme aromatase in certain target cells are known to be mediated by the androgen receptor. The androgen receptor are coded by a single copy gene, which is located on the X chromosome. The functional relevance of the segmented domain structure of the NR superfamily is supported by the results of deletion mutagenesis and domain swapping among various receptor proteins. The interaction between the amino terminal and the steroid-binding carboxy-terminal end in the AR transactivation function was initially indicated by the finding that a segment within the hormone-binding domain exerts an inhibitory influence in the transcription regulatory activity of the AR, and the deletion of this region results in ligand-independent activation of the receptor.

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.

Age-dependent increase of heme oxygenase–1 gene expression in the liver mediated by NFκB

Heme, the iron-porphyrin coordination complex, released from the degradation of hemoproteins, is a strong prooxidant. It is enzymatically degraded by heme oxygenase to free iron, carbon monoxide and biliverdin. Biliverdin and its reduced metabolite bilirubin are two potent physiological antioxidants. Here we show a progressive increase of steady-state levels of the mRNA encoding the inducible isoform of this enzyme (heme oxygenase-1) in the rat liver during aging. We had previously reported that aging is associated with increased activation of the nuclear factor kappaB (NFkappaB). We now provide evidence to establish that overexpression of NFkappaB in transfected liver-derived HepG2 cells can cause a marked induction of the endogenous heme oxygenase-1 (HO-1) mRNA and activation of the cotransfected HO-1 gene promoter. Taken together, these results support the conclusion that enhanced oxidative stress during aging is accompanied by compensatory induction of the antioxidant enzyme HO-1 through activation of the NFkappaB pathway.

Impacts of transcriptional regulation on aging and senescence

The genetic makeup of the organism appears to dictate the species-specific rate of aging and the maximum life-span potential. The genotype is converted to phenotype through transcriptional and translational regulation. A group of gene regulatory proteins (transcription factors) play critical roles in controlling the rates of transcription of specific genes by directly interacting with regulatory sequences at gene promoters. Here, we review the basic mechanism of transcriptional control and the role of a number of transcription factors whose level and/or activity alter with age. Among these age-dependent transcription factors, many are involved in the regulation of stress and inflammatory responses and are subjected to functional alterations by reactive oxygen species (ROSs). A progressive rise of oxidative stress, impaired ability to cope with stressful stimuli and prolongation of the inflammatory response are some of the hallmarks of the senescent phenotype. Results published to date are supportive of the concept that a species-specific program of the temporal regulation of genes with additional modulation by a number of epigenetic factors, mediates the age-dependent deterioration of physiological functions and development of the senescent phenotype.

Lysine methylation and functional modulation of androgen receptor by Set9 methyltransferase.

Lysine methyltransferases modulate activities of transcription factors and transcription coregulators by methylating specific lysine residue(s). We report that the androgen receptor (AR) is methylated at lysine-630 by Set9, which was originally identified as a histone H3K4 monomethyltransferase. Alanine substitution of lysine-630 prevented AR methylation in vitro and in vivo. Set9 methylated the nuclear and cytoplasmic AR utilizing the cofactor S-adenosyl-methionine. A pan-methyllysine antibody recognized endogenous AR, and Set9 coimmunoprecipitated with nuclear and cytoplasmic AR. Set9 overexpression potentiated AR-mediated transactivation of the probasin promoter, whereas Set9 depletion inhibited AR activity and target gene expression. Similar to AR, chromatin occupancy of Set9 at androgen response elements (AREs) was androgen dependent, and associated with methylated histone H3K4 chromatin activation marks and p300/CBP associated factor acetyltransferase recruitment. Set9 depletion increased the histone H3K9-dimethyl repressive mark at AREs and reduced histone activation marks and occupancy of p300/CBP associated factor. K630A mutation reduced amino- and carboxy-terminal (N-C) interaction in Set9-intact cells, whereas N-C interaction for wild-type AR was reduced upon Set9 depletion. The K630A mutant was resistant to loss of activity from Set9 silencing and to increase of activity from Set9 overexpression. The K630 dependence of Set9-regulated N-C interaction and AR activity suggests that Set9 directly acts on AR at the amino acid level. Chromatin recruitment of Set9 to AREs is suggestive of its additional role as a transcriptional coactivator. Because the cellular metabolic state determines the level of S-adenosylmethionine and consequently the activity of Set9, the enhanced activity of methylated AR may have special significance in certain metabolic contexts.

Vitamin D receptor regulation of the steroid/ bile acid sulfotransferase SULT2A1

SULT2A1 is a sulfo-conjugating phase II enzyme expressed at very high levels in the liver and intestine, the two major first-pass metabolic tissues, and in the steroidogenic adrenal tissue. SULT2A1 acts preferentially on the hydroxysteroids dehydroepiandrosterone, testosterone/dihydrotestosterone, and pregnenolone and on cholesterol-derived amphipathic sterol bile acids. Several therapeutic drugs and other xenobiotics, which include xenoestrogens, are also sulfonated by this cytosolic steroid/bile acid sulfotransferase. Nonsteroid nuclear receptors with key roles in the metabolism and detoxification of endobiotics and xenobiotics, such as bile acid-activated farnesoid X receptor, xenobiotic-activated pregnane X receptor and constitutive androstane receptor, and lipid-activated peroxisome proliferator-activated receptor-alpha, mediate transcription induction of SULT2A1 in the enterohepatic system. The ligand-activated vitamin D receptor (VDR) is another nuclear receptor that stimulates SULT2A1 transcription, and the regulatory elements in human, mouse, and rat promoters directing this induction have been characterized. Given that bile acid sulfonation is catalyzed exclusively by SULT2A1 and that the 3alpha-sulfate of the highly toxic lithocholic acid is a major excretory metabolite in humans, we speculate that a role for the VDR pathway in SULT2A1 expression may have emerged to shield first-pass tissues from the cytotoxic effects of a bile acid overload arising from disrupted sterol homeostasis triggered by endogenous and exogenous factors.

Calorie restriction delays age-dependent loss in androgen responsiveness of the rat liver.

We have shown that restricted calorie intake retards age‐associated loss in androgen responsiveness of the rat liver. Sustained androgen receptivity delays age‐dependent decline in the synthesis of the androgen‐inducible α2u globulin and derepression of the androgen‐repressible senescence marker protein (SMP‐2). Quantitation of mRNAs for α2u globulin and SMP‐2 in the liver of animals of various ages maintained on either ad libitum or restricted diets revealed that, although the 27‐month‐old ad libitum‐fed rat had only 5% as much α2u mRNA as the 6‐month‐old rat; the mRNA level was as high as 45% in the 27‐month‐old food‐restricted rat. Conversely, the 27‐month‐old food‐restricted rat had a much reduced amount (45%) of SMP‐2 mRNA compared to the age‐matched control that was allowed unlimited access to food. Furthermore, we have correlated the effect of dietary restriction on age‐dependent changes in specific gene expression with the hepatic level of the immunoreactive cytoplasmic androgen‐binding (CAB) protein. We observed that senescence in the male causes a substantial decrease in the circulating level of testosterone. However, dietary restriction does not retard the rate of decline in the plasma level of the male hormone during aging. These results indicate that age‐dependent changes in the expression of androgen‐responsive genes (α2u globulin and SMP‐2) reflect changing androgen sensitivity and that food restriction may directly influence the androgen receptivity of the liver.— Chatterjee, B.; Fernandes, G.; Yu, B. P.; Song, C.; Kim, J. M.; Demyan, W.; Roy, A. K. Calorie restriction delays age‐dependent loss in androgen responsiveness of the rat liver. FASEB J. 3: 169‐173; 1989.

Gene regulation for the senescence marker protein DHEA-sulfotransferase by the xenobiotic-activated nuclear pregnane X receptor (PXR)

Dehydroepiandrosterone (DHEA)-sulfotransferase (SULT2A1) is a phase II metabolizing/detoxifying enzyme with substrate preference for physiological hydroxysteroids, diverse drugs and other xenobiotics. The first-pass tissues (liver and intestine) express SULT2A1 at high levels. In senescent male rodents, Sult2A1 gene transcription in the liver is markedly enhanced and calorie restriction retards this increase. Age-associated loss of the liver expression of androgen receptor in part explains the up-regulation of Sult2A1 expression at late life, since androgen receptor is a negative regulator of this gene. In line with its role in xenobiotic metabolism, the Sult2A1 gene is induced by the pregnane X receptor (PXR). PXR is a xenosensing nuclear receptor that is activated by endobiotic (natural steroids) and xenobiotic (therapeutic drugs and environmental chemicals) molecules. An inverted-repeat arrangement (IR0) of the consensus half site binding sequence for nuclear receptors mediates the xenobiotic induction of the Sult2A1 promoter. The IR0 element is a specific binding site for PXR and its heterodimer partner retinoid X receptor (RXR-alpha) and it directs PXR-mediated induction of a heterologous promoter. In contrast to the loss of androgen receptor expression, PXR and RXR-alpha mRNA expression is invariant during aging. Repression by the androgen receptor and induction by PXR may act coordinately to cause the senescence associated and xenobiotic mediated stimulation of Sult2A1 transcription. Increased Sult2A1 expression appears to be an adaptive response to ensure optimal metabolism of Sult2A1 substrates at old age.

Loss of androgen receptor in aging and oxidative stress through Myb protooncoprotein-regulated reciprocal chromatin dynamics of p53 and poly(ADP-ribose) polymerase PARP-1.

Poly(ADP-ribosyl)ation of transcription factors and coregulators, mediated by the poly(ADP-ribose) polymerase PARP-1, has been emerging as an important epigenetic mechanism that controls transcriptional dynamics in response to diverse intra- and extracellular signals. PARP-1 activity is also implicated in the regulation of mammalian lifespan. Herein we show that transcriptional down-regulation of androgen receptor (AR) in the aging rat liver and in oxidatively stressed hepatoma cells involves exchange of a PARP-1-associated, p/CAF-containing coactivator assembly for a p53-interacting, Groucho/TLE1-, and mSin3A-included corepressor complex at an age- and oxidant-responsive DNA element (age-dependent factor (ADF) element) in the AR promoter. The coregulator switch is mediated by B-Myb and c-Myb, which bind to the ADF element and physically associate with PARP-1 and the tumor suppressor p53. Heterogeneous nuclear ribonucleoprotein K, residing at the ADF element in association with PARP-1, may serve a platform role in stabilizing the activating complex. PARP-1 coactivated B-Myb- and c-Myb-mediated transactivation of the AR promoter, and p53 antagonized the B-Myb/c-Myb-induced AR promoter activation. PARP-1, heterogeneous nuclear ribonucleoprotein K, B-Myb, and c-Myb each serves as a positive regulator of cellular AR content, whereas p53 negatively regulates AR expression. Our results identify a shared, PARP-1-regulated sensing mechanism that coordinates transcriptional repression of AR during aging and in response to oxidative stress. This study may provide insights as to how advancing age and intracellular redox balance might influence androgen-regulated physiology.

Androgen and estrogen sulfotransferases of the rat liver: physiological function, molecular cloning, and in vitro expression.

Cloning of the androgen and estrogen sulfotransferases has allowed studies on their spatiotemporal regulation and physiological function. These two enzymes appear to be involved in regulating target cell sensitivity for the androgenic and estrogenic steroids. Recombinant androgen sulfotransferase produced in insect Sf9 cells through a baculoviral expression vector is capable of transferring the sulfate from PAPS (phosphoadenosine phosphosulfate) to 3 alpha, 3 beta, 17 alpha, and 17 beta hydroxyl groups of a number of steroid substrates indicating a broad range of substrate specificity.